The complete primary structure of LDH-A4 isozymes from human and mouse has been determined by sequence analyses of LDH-A cDNA and protein. The structural relationship of cancer-associated LDH-K to LDH-A isozyme has been illustrated. Partial amino acid sequences of LDH-B4 isozymes from human and mouse have also been determined by amino acid sequencing. The amino acid sequences of 100% of the 330 residues from mouse testicular LDH-C4 and 84% of rate LDH-C4 have been determined. Sequence comparison among mammalian LDH isozymes clearly indicates that A4 (muscle) and B4 (heart) isozymes shows a closer evolutionary relationship to each other than either to the C4 (testis) isozyme. Recently, LDH-A4 isozymes have been found to be single-stranded DNA binding proteins which may play important roles in DNA replication, repair and recombination. The relationship of the protein structure to multiple functions as enzyme and as DNA binding protein will be further studied by in vitro directed mutagenesis.